PROJECT SUMMARY: Recurrent laryngeal nerve (RLN) injury occurs in tens of thousands of patients per year in the United States. It results in synkinetic reinnervation inducing vocal fold paralysis and severe dysphonia. This produces significant patient morbidity as the voice is a unique and integral part of each person's identity, impacting virtually every aspect of our daily lives, from speaking to loved ones, to engaging in business or simply completing daily tasks. Voice dysfunction leads to fear and isolation, as well as impaired quality of life, disability claims and lost worker productivity similar to severe chronic diseases, resulting in a significant societal burden. Current therapies are suboptimal and there are no treatments that can restore vocal fold motion and normal voice. There are critical gaps in our knowledge about the molecular mechanisms underlying axonal guidance during nerve regeneration. Our long-term goals are to identify the expression and actions of guidance cues and to manipulate them to guide selective, non-synkinetic reinnervation of the laryngeal muscles after RLN injury, with restoration of normal vocal fold function and normal voice. Our previous work has revealed that in both embryologic development and post-RLN injury, laryngeal muscles are innervated in similar sequences. Yet, post-RLN injury reinnervation is disordered, resulting in synkinesis with vocal fold dysfunction. Our laboratory has identified Netrin-1 and GDNF as the factors most likely to play a central role in axon guidance during RLN regeneration. We have shown that their expression in laryngeal muscles and the expression of their receptors in the nucleus ambiguus (NA) are chronologically coordinated with axonal reinnervation. We have demonstrated that manipulation of these guidance cues can alter the pattern of reinnervation. These findings suggest a controlled but dysfunctional expression of guidance cues resulting in synkinetic reinnervation and vocal fold paralysis after RLN injury. In comparison, developmental innervation, which is also tightly controlled chronologically, results in normal vocal fold function. Our central hypothesis is that Netrin-1 and GDNF are critical guidance cues in laryngeal reinnervation. Further, manipulation of their pathways post-RLN injury to mimic those seen during normal development will in result in selective, non- synkinetic reinnervation with restoration of normal vocal fold function. Guided by strong preliminary data, we seek to pursue the following three Specific Aims: 1) to describe the patterns of expression of GDNF, Netrin-1, and their receptors during innervation of the larynx in development 2) to describe the pathways that modulate the function of GDNF, Netrin-1 and their receptors during innervation in development and during reinnervation post-RLN injury and 3) to evaluate in vitro the effects of modulating pathways in the NA and muscle. This research will broadly impact the field of nerve regeneration, advancing our knowledge of the mechanisms of axonal pathfinding with the potential to transform the treatment of vocal fold paralysis.